We show for the first time that expression of mutant Huntington's disease protein (mhtt) functionally inhibits vesicular trafficking and endocytosis in primary striatal neurons from mice. Unexpectedly, mhtt does not inhibit clathrin-mediated endocytosis as would be predicted based on known htt interacting partners. Instead, mhtt inhibits a non-clathrin, caveolar pathway at the step of internalization. A second surprise was that inhibition of caveolar endocytosis was accompanied by an unforeseen and striking accumulation of intracellular cholesterol in primary neurons. Cholesterol accumulation also occurred in cell lines induced to express mhtt, and in transgenic animals in vivo. These data not only provide the first direct link between mhtt and aberrant caveolar endocytosis. But also suggest a novel mechanism for Huntington's Disease (HD) neurotoxicity in which cholesterol metabolism is perturbed. Abnormal cholesterol accumulation has also been implicated in Alzheimer's (AD), Niemann-Pick Type C (NPC) and other lipid storage diseases. However, the significance of these observations was not previously known. Prusiner and colleagues have raised the possibility that non-classical caveolar endocytosis may be the site of misfolding of PrP protein and conversion to the pathogenic form. For the first time, we provide in vitro and in vivo evidence that intracellular cholesteroraccumulation via caveolae occurs in mhtt-expressing animals and correlates with HD pathophysiology. Disturbance in cholesterol homeostasis or trafficking may also be a common element in neurotoxicity via a caveolar-related mechanism. In this proposal, we test the mechanism by which mhtt might impair trafficking of cholesterol to cause disease, and the role of caveolin in these abnormal processes. Using a series of transgenic models, we will directly test whether an interaction between mhtt and caveolin is responsible for cholesterol accumulation and pathophysiology in vivo. Primary neurons are isolated from these animals and we will test whether caveolae, mhtt and cholesterol accumulate at the same subcellular site. Using real-time imaging we will test three likely mechanisms by which cholesterol accumulation arises: Mhtt may cause (1) mislocalization of cholesterol preventing normal efflux or recycling, (2) an increase in cholesterol internalization via LDL from glia, or (3) an over stimulation of cholesterol biosynthesis in neurons to compensate for a trafficking defect. Finally, we will dissect the molecular mechanism by which interaction of cav-1 and mhtt causes the trafficking defects by mutating functional regions of cav-1 and mhtt. These results will establish not only the mechanisms of toxicity in mhtt expressing animals but also test whether these effects are common to other disorders. Further, the data will shed light on a poorly understood pathway of endocytosis that may be involved in the defects.